TABLE 3.

Steady-state kinetic parameters of wild-type Δ¹-KSTD1 and its mutants

The dehydrogenase activity was measured using seven different 4-AD substrate concentrations ranging from 7.5 to 240, 60 to 1,000, and 40 to 700 μm for wild-type, Y119F, and Y487F enzyme, respectively. The enzyme concentrations were approximately 360 × 10⁻⁶ μm, 1.07 μm, and 18 × 10⁻³ μm for the wild-type, Y119F, and Y487F enzyme, respectively. The values are the averages of triplicate measurements.

Variant	Specific activity	Activity	Km	kcat	kcat/Km
	nmol·mg−1·min−1	%	μm	s−1	s−1·m−1
Wild-type	72,900 ± 1,500	100	37.0 ± 0.8	68.1 ± 1.4	1,838,000 ± 67,000
Y119F	38.6 ± 2.5	0.05	335 ± 51	0.036 ± 0.002	108 ± 12
Y318F	NMa	NM	NM	NM	NM
Y487F	1,868 ± 53	2.6	193 ± 12	1.7 ± 0.05	9,061 ± 844

^a NM, not measurable.